Qdot ITK Carboxyl Quantum Dots

Transcription

1 Qdot ITK Carboxyl Quantum Dots Catalog nos. Q21341MP, Q21391MP, Q21331MP, Q21311MP, Q21301MP, A10200, Q21321MP, Q21361MP, Q21371MP Table 1. Contents and storage information. Material Amount Concentration Storage Stability Qdot ITK carboxyl quantum dots 250 µl 8 µm solution in 50 mm borate, ph C Do not freeze When stored as directed the product is stable for at least 6 months. Approximate fluorescence excitation/emission spectra: See Figure 2. Introduction Structure of Qdot Nanocrystals Qdot ITK (Innovator s Tool Kit) carboxyl quantum dots are made from nanometerscale crystals of a semiconductor material (CdSe), which are shelled with an additional semiconductor layer (ZnS) to improve their chemical and optical properties. The Qdot 705 and Qdot 800, which include CdSeTe, are made in a similar fashion. These materials have narrow, symmetric emission bands with emission maxima near 525 nm, 565 nm, 585 nm, 605 nm, 625 nm, 655 nm, 705 nm, or 800 nm. This core-shell material (Figure 1A) is further coated with a polymer layer that allows facile dispersion of the quantum dots in aqueous solutions with retention of their optical properties. The polymer coating has COO - surface groups available for modifications such as macromolecule attachment (Figure 1B). Qdot ITK carboxyl quantum dots are about the size of a large macromolecule or protein. Figure 1. A. Transmission electron microscope image of core-shell Qdot nanoparticles at 200,000x magnification. Scale bar = 20 nm. B. Schematic of the overall structure of a Qdot conjugate. The layers represent the distinct structural elements of the Qdot nanocrystal conjugates, and are roughly to scale. Revised: 20 December 2007 MP 19020

2 Optical Properties The optical properties of Qdot are different from those of typical organic dye molecules. The colors of light that Qdot emit are strongly dependent on particle size, creating a common platform of fluorescent labels emitting from green to the near IR, all manufactured from the same underlying semiconductor material (see Bibliography, references 1 11 in the Appendix). The size of Qdot is tightly controlled in the production process, resulting in materials with narrow and symmetric emission bands and that are extremely bright and photostable. While the fluorescence emission bands from the Qdot 705 and Qdot 800 are broader than the emission bands of the visible wavelength Qdot, all the Qdot fluorophores have strong emission intensity and superior photostability. Note that Qdot 705 and 800 nm emissions cannot be seen by eye, but are easily detected by many cameras and detectors. These properties are exploited in a variety of immunofluorescence techniques, and can result in substantially better results than are attainable with conventional fluorescent labels (see Bibliography, references 12 18). Though these materials are compatible with a number of standard fluorescence techniques, there are some novel aspects of their chemistry and detection that require careful consideration to obtain optimal results. Spectral Characteristics Organic fluorescent dyes have excitation and emission spectra with a relatively small Stokes shift, which means that the optimal excitation wavelength is close to the emission peak. Filter sets used with fluorescent dyes reflect this characteristic. 19 Light absorption efficiency of Qdot increases dramatically to the blue of the emission (Figure 2). These unique spectral properties are due to the semiconductor material that makes up the core of the Qdot, which gives rise to both their absorption and emission properties. (see Bibliography, references 1 11). Despite their broad wavelength range of light absorption, the emission wavelength of these materials is independent of the excitation wavelength. For example, whether exciting at 400 nm or 633 nm, the shape of the emission band of Qdot 655 remains the same, while the intensity is approximately 11-fold higher with 400 nm excitation. Light absorption and consequent excitation at shorter wavelength, with fixed emission, results in a large apparent Stokes shift. Short wavelength excitation improves sensitivity by reducing autofluorescence and takes advantage of the inherently greater light absorption of these materials in the blue to violet spectral region, greatly simplifying simultaneous, multiplexed detection of several Qdot nanocrystal colors. See Appendix 3 for extinction coefficients of the different materials at common excitation wavelengths. Optical Filter Selection To achieve the optimal signal from Qdot conjugates, we recommend using Qdot optimized filter sets that are available from Omega Optical, Semrock, or Chroma Technology Corporation (see Appendix 2 for details). Qdot conjugates can also be viewed through some standard filter sets, albeit with lower detection efficiency and reduced brightness. For example, three Omega Optical standard filter sets capable of detecting Qdot 705 conjugates are XF140-2 (Alexa Fluor 633 & Alexa Fluor 647), XF70 (Alexa Fluor 660 & Cy5), and XF141-2 (Cy5.5). Visualization of Qdot conjugates using a custom filter set is preferred because excitation and detection is less efficient using filters that have not been selected specifically for use with Qdot conjugates. Using a custom filter set, Qdot 605 conjugate signal is approximately five times as bright as it is using the TRITC filter set, and approximately ten times brighter than it is using the Texas Red /Cy3.5 filter set (Figure 3). Qdot optimal filters and standard filter sets are available from different filter manufacturers. Appendix 2 illustrates some common filter sets and the optimal filter set recommendations for the available Qdot conjugates. Use of the optimal filter set is critical for attaining optimal signal and sensitivity in your experiments. Qdot ITK Carboxyl Quantum Dots 2

3 Figure 2. Typical absorption and emission spectra of Qdot 525 conjugate (1), Qdot 565 conjugate (2), Qdot 585 conjugate (3), Qdot 605 conjugate (4), Qdot 625 conjugate (5), Qdot 655 conjugate (6), Qdot 705 conjugate (7), Qdot 800 conjugate (8). Before You Begin Qdot have chemical and optical properties that provide significant advantages over conventional fluorophores in both sensitivity and stability in fluorescent labeling and tracking applications. Qdot ITK carboxyl quantum dots are used in a wide variety of labeling and tracking applications, including preparation and use of peptide derivatives, 20 nucleic acid conjugates, 21,22 polysaccharide conjugates, 23 in stem cell tracking 24 and for other uses in which ultrabright and stable fluorescence is desired. Figure 3. Detection of Qdot conjugates on tissue sections with recommended and standard filter sets. Mouse kidney sections were stained with Qdot 605 conjugate, and then images were collected on a Nikon epi-fluorescence microscope in 16 bit capture mode. The mean fluorescence of positively stained samples was extracted using Scion Image software. The recommended Qdot filter set included a 460 nm short pass exciter, a 475 nm dichroic, and a 605/20 nm band pass emitter. The Cy3 filter set included a 545/30 nm exciter, a 570 nm dichroic, and a 610/75 nm emitter. The Texas Red filter set included a 560/40 nm exciter, a 595 nm dichroic, and a 630/60 nm emitter. Qdot ITK Carboxyl Quantum Dots 3

4 For additional applications such as immunocytochemistry, tissue section staining, western blotting, as well as multiplexing using Qdot streptavidin and secondary antibody conjugates, download the Qdot Conjugates Protocol Handbook from For additional information and useful protocols for various applications with Qdot and their conjugates, see Quantum Dots: Applications in Biology (Methods in Molecular Biology). 25 Note: The near infrared 705 and 800 nm quantum dot emissions cannot be seen by eye, but are easily detected by many cameras and detectors. General Considerations Buffer compatibility In our experience, Qdot ITK carboxyl quantum dots and many of their conjugates have stable emission in a number of buffers, and the quantum yield and colloidal dispersion of conjugates made with these materials has been found to be stable at physiological and nearphysiological ph (not investigated outside this range) in Tris, HEPES, phosphate, and borate buffers. In addition, a number of surfactants and additives such as Tween 20, Triton X-100, and EDTA, among others, have been shown to maintain nanocrystal fluorescence when used at up to 0.5% concentration. We recommend storage of the Qdot ITK carboxyl quantum dot product at the concentration at which it is shipped, rather than at high dilution. Storage of Qdot ITK carboxyl quantum dots and their macromolecule conjugates at working dilution may result in substantial performance degradation. While we have not characterized the stability of all Qdot ITK carboxyl quantum dots under all of these conditions, we anticipate similar levels of stability across the range of product colors. Qdot nanocrystal toxicity We have not investigated the toxicity of Qdot ITK carboxyl quantum dots. The materials are provided in a solution which is ~2 mm total Cd concentration; however, the CdSe core is encapsulated in a crystalline shell of ZnS and the amphophilic polymer coating, which may help prevent formation of free Cd. We have demonstrated the utility of these materials in a variety of live-cell in vitro labeling experiments, but do not have systematic data on the toxicity of the materials to humans, to animals, or to cells in culture. FRET or close-proximity quenching We have not systematically investigated the energy transfer properties of the Qdot, though they may have useful properties as energy transfer donors and acceptors. We have investigated the fluorescence of Qdot 605 conjugates which are coupled to each other through a bis-biotin linker, and found that the emission intensity of the materials was unperturbed at any concentration of biotin cross-linker. These results suggest that the interparticle quenching of these Qdot conjugates is negligible. Published literature indicates that Qdot can be used as energy acceptors in time-resolved FRET (TR-FRET) studies. 26 Disposal of Qdot Conjugate The Qdot conjugate contains cadmium and selenium in an inorganic crystalline form. Dispose of the material in compliance with all applicable local, state, and federal regulations for disposal of these classes of material. For more information on the composition of these materials, consult the Material Safety Data Sheet. Conjugating Qdot ITK carboxyl quantum dots A protocol for conjugating streptavidin to Qdot ITK carboxyl quantum dots is supplied below. You can use similar methods to conjugate other proteins and biomolecules of interest, providing they are compatible with the coupling chemistry described below or with alternate conjugation chemistry under consideration. The amounts suggested below may need to be adjusted to accomodate your conjugation requirements. Qdot ITK Carboxyl Quantum Dots 4

5 Conjugation of Qdot ITK Carboxyl Quantum Dots to Steptavidin Materials Required but Not Provided 10 mg N-ethyl-N -dimethylaminopropyl-carbodiimide (EDC) 10 mg/ml Streptavidin (Invitrogen Cat. no. S888) in 10 mm borate buffer, ph mm borate buffer, ph mm borate buffer, ph 8.3 Ultrafiltration units with 100 kda cutoff, size 4 ml (Amicon Ultra-4 Millipore Cat. no. UFC810008) or size 15 ml (Amicon Ultra 15 Millipore Cat. no. UFC910008) Filter syringes: Acrodisc 25 mm PF Syringe Filter with 0.8/0.2 µm Supor Membrane or Acrodisc Syringe Filter 0.2 µm Supor Membrane Low Protein Binding Non Pyrogenic PES (polyethersulfone) syringe filters, 0.2 µm (Whatman Cat. no ) Preparing Streptavidin Solution Prepare a 10 mg/ml streptavidin solution in 10 mm borate buffer, ph 7.4. Mix well. You will need 80 nmol of streptavidin for the conjugation reaction. Amount The molar concentration of each reagent used during the conjugation protocol is described below. Use these concentrations for the initial experiments and based on your results, you may need to optimize these concentrations to obtain the desired level of conjugation. Reagent Concentration Equivalent Qdot Reagent 2 nmol (250 µl at 8 µm concentration) 1 Streptavidin 80 nmol ( mg at 10 mg/ml) 40 EDC 3000 nmol (0.57 mg at 10 mg/ml) 1500 Experimental Protocol Conjugation Protocol Please read the entire protocol before starting. 1.1 In a small glass vial with a small stirbar, dilute 250 µl of 8 µm stock solution of Qdot ITK carboxyl quantum dots to 2 ml using 10 mm borate buffer, ph 7.4. Mix well by stirring. 1.2 Add 0.48 ml of 10 mg/ml streptavidin to the Qdot ITK carboxyl quantum dots reagent (step 1.1). Continue stirring. 1.3 Weigh ~5 mg of EDC in a 1.5 ml microcentrifuge tube and add 0.5 ml deionized water to obtain a 10 mg/ml EDC stock solution. Prepare EDC solution just before use. 1.4 Immediately, add 57 µl of 10 mg/ml EDC stock solution to the Qdot solution (step 1.2). 1.5 Stir gently for 1 2 hours at room temperature for the conjugation. 1.6 Filter the conjugate solution through a 0.2 µm PES syringe filter to remove any large aggregates and transfer the solution to a clean centrifugal ultrafiltration unit (100 kda cutoff). 1.7 Centrifuge at the recommended speed for the ultrafiltration unit for at least 5 buffer exchanges using 50 mm borate buffer, ph 8.3 to remove any excess unbound protein. Ensure Qdot ITK Carboxyl Quantum Dots 5

6 that the volume of concentration is >10-fold (e.g., 4 ml to <400 µl) each time. 1.8 After ultracentrifugation is complete, filter the solution through a 0.2 µm syringe filter or a 0.8/0.2 µm combination syringe filter to remove any aggregates. Store the Qdot conjugate solution at 4 C. Do not freeze the nanocrystal conjugate. Appendix 1: Troubleshooting Guide The properties of Qdot conjugates are different from fluorescent dyes and may require slight modifications to current protocols. We ve included this section to help with some specific issues that may arise while using these materials. No Signal Optical Setup suitability Make sure that you are using an appropriate filter set to detect the signals. See Appendix 2 for a list of appropriate and optimal filters for the Qdot conjugates. Contact Technical Support (probestech@invitrogen.com) for more details on particular filter set requirements. Qdot conjugate luminosity Qdot conjugates normally fluoresce brightly under a hand-held ultraviolet lamp (long wave, such as the type used to visualize ethidium bromide on agarose gels). The 705 and 800 nm quantum dot emission cannot be seen by eye, but is detected by many cameras and detectors. Though we have not seen pronounced loss of fluorescence of these materials under any storage conditions that we have investigated, we have not been able to examine all storage conditions. If the Qdot conjugates do not appear to fluoresce under the long wave UV excitation, contact Technical Support (probestech@invitrogen.com) for assistance. Qdot ITK Carboxyl Quantum Dots 6

7 Appendix 2: Optimal Usable Filter Sets for Qdot Conjugates Table 3. Omega optical filter set for Qdot conjugates * 800 * Color Optimal filter sets Usable filter sets All colors XF301 Qdot 525 filter set Dichroic: 475DCLP Emitter: 525WB20) XF302 Qdot 565 filter set Emitter 565WB20) XF303 Qdot 585 filter set (Exciter: 1 425DF45 or Emitter: 585WB20) XF304 Qdot 605 filter set Emitter: 605WB20) XF305 Qdot 655 filter set Emitter: 655WB20) XF306 Qdot 705 filter set Emitter: 710AF40) XF307 Qdot 800 filter set Emitter: 800WB80) XF300 Qdot filter set Dichroic: 475DCLP Emitters: 800WB80, 840WB80, 710AF40, 655WB20, 605WB20, 585WB20, 565WB20, and 525WB20) XF100-3, XF100-2, XF115-2, XF89-2 XF104-2, XF105-2 XF101-2, XF137-2, XF152-2 XF108-2, XF102-2, XF103-2 XF102-2, XF40-2, XF42, XF45 XF140-2, XF70, XF110-2, XF141-2, XF48-2 XF308 Qdot 800 filter set for multiplexing Emitter: 840WB80) XF129-2, XF130-2 *The 705 and 800 nm quantum dot emission cannot be seen by eye and must be detected with an IR-sensitive detector. For viewing multiple colors of Qdot through microscope eyepieces. Qdot ITK Carboxyl Quantum Dots 7

8 Table 4. Semrock filter sets for Qdot conjugates. 525 Color Optimal filter sets Usable filter sets BrightLine QD525-A Filter Sets: QD525-A-000 or QD525-A-000-ZERO (Exciter: FF01-435/40-25) (Dichroic: FF510-Di01-25 x 36) (Emitter: FF01-525/15-25) GFP-3035B FITC-3504B or YFP-2427A TRITC-A BrightLine QD605-A Filter Sets: QD605-A-000 or QD605-A-000-ZERO (Exciter: FF01-435/40-25) (Dichroic: FF510-Di01-25 x 36) (Emitter: FF01-605/15-25) BrightLine QD625-A Filter Sets: QD625-A-000 or QD625-A-000-ZERO (Exciter: FF01-435/40-25) (Dichroic: FF510-Di01-25 x 36) (Emitter: FF01-625/15-25) BrightLine QD655-A Filter Sets: QD655-A-000 or QD655-A-000-ZERO (Exciter: FF01-435/40-25) (Dichroic: FF510-Di01-25 x 36) (Emitter: FF01-655/15-25) TRITC-A Texas Red (4040B) Texas Red (4040B) 705* -- Cy5-4040A or Cy5.5-A 800* -- Cy7-A LP multi QDLP-A Filter Set: QDLP-A-000 (Exciter: FF01-435/40-25) (Dichroic: FF510-Di01-25 x 36) (Emitter: FF01-500/LP-25) CFW-LP01-CLINICAL *The 705 and 800 nm quantum dot emission cannot be seen by eye and must be detected with an IR-sensitive detector. For viewing multiple colors of Qdot through microscope eyepieces. Qdot ITK Carboxyl Quantum Dots 8

9 Table 5. Chroma Technology filter sets for Qdot conjugates * 800* Color Optimal filter sets Usable filter sets All colors Qdot 525 filter set (20 nm EM; 32006) (460SPUV/475DCXRU/D525/20nm) Qdot 525 filter set (40 nm EM; 32010) (460SPUV/475DCXRU/D525/40nm) Qdot 565 filter set (20 nm EM; 32005) (460SPUV/475DCXRU/D565/20nm) Qdot 565 filter set (40 nm EM; 32009) (460SPUV/475DCXRU/D565/40nm) Qdot 585 filter set (20 nm EM; 32004) (460SPUV/475DCXRU/D585/20nm) Qdot 585 filter set (40 nm EM; 32008) (460SPUV/475DCXRU/D585/40nm) Qdot 605 filter set (20 nm EM; 32003) (460SPUV/475DCXRU/D605/20nm) Qdot 605 filter set (40 nm EM; 32007) (460SPUV/475DCXRU/D605/40nm) Qdot 655 filter set (20 nm EM; 32011) (460SPUV/475DCXRU/D655/20nm) Qdot 655 filter set (40 nm EM; 32012) (460SPUV/475DCXRU/D655/40nm) Qdot 705 filter set (20 nm EM; 32014) (460SPUV/475DCXRU/D705/20nm) Qdot 705 filter set (40 nm EM; 32015) (460SPUV/475DCXRU/D705/40nm) Qdot 800 filter set (30 nm EM; 32020) (460SPUV/475DCXRU/D800/30nm) Qdot 800 filter set (50 nm EM; 32021) (460SPUV/475DCXRU/D800/50nm) Qdot Multiple Emission Set (71014) (460SPUV, 475DCXRU, D525/20nm, D605/20nm, D565/20nm, D585/20nm) FITC/RSGFP/Bodipy /Fluo-3/DiO (41001), FITC/RSGFP Longpass (40012), BFP to GFP FRET (31032), BFP to GFP FRET wide excitation (31034), GFP wide blue excitation (31054) Eosin (41011), Cascade Yellow (31038), JP2(YGFP with EGFP-31040, Auramine (31015) R-PE (41003), Rhodamine LP (41032, FITC/PI (41016) Cy3 narrow excitation (41007a), Texas Red /Cy3.5 (31004), TRITC (41002, 41002a, 41002b), Ethidium Bromide (41006) Texas Red (41004), Propidium Iodide (41005), Fura Red (31012), Chlorophyll (31017), Allophycocyanin (31006) Cy5 Longpass (41024), Cy5 (41008), Cy5 narrow excitation (41033), Cy5.5 (41023), Alexa Fluor 680 (41042), Cy5.5 (red-shifted; 41022) Cy7 (41009), Li-Cor for IRDye 800 (41037), Cy7 (SP106) UV (11000V2), Blue/Violet (11003V2), UV/Violet (11011V2) *The 705 and 800 nm quantum dot emission cannot be seen by eye and must be detected with an IR-sensitive detector. For viewing multiple colors of Qdot through microscope eyepieces. Qdot ITK Carboxyl Quantum Dots 9

10 Appendix 3: Extinction Coefficients Table 6. Extinction coefficients of Qdot conjugates at common excitation wavelengths. Product Qdot 525 Qdot 565 Qdot 585 Qdot 605 Qdot 625 Qdot 655 Qdot 705 Qdot nm, in cm 1 M nm, in cm 1 M nm, in cm 1 M nm, in cm 1 M 1 710, , ,000 Not applicable 1,900,000 1,100, , ,000 3,500,000 2,200, , ,000 4,400,000 2,800,000 1,100, ,000 14,700,000 9,900,000 2,700, ,000 9,100,000 5,700,000 2,900,000 2,100,000 12,900,000 8,300,000 3,000,000 2,100,000 12,600,000 8,000,000 3,000,000 2,000,000 Appendix 4: Bibliography There are a number of references that describe the size-dependent properties of the semiconductor. These range in complexity from fairly straightforward descriptions to fairly comprehensive mathematical and physical descriptions of the optical properties. In addition, we have included some representative references that describe the core-shell structures, and the improved chemical properties that are obtained through such structures. References 8 11 describe quantum dots and FRET: 1. Sci Am 285, 66 (2001); 2. J Phys Chem B 100, (1996); 3. J Am Chem Soc 115, 8706 (1993); 4. Phys Rev B 53, (1996); 5. J Phys Chem 100, 468 (1996); 6. J Phys Chem B. 101, 9463 (1997); 7. J Am Chem Soc 119, 7019 (1997); 8. Nano Lett 1, 469 (2001); 9. J. Am. Chem. Soc 126, 301 (2004); 10. Nat Mater 2, 630 (2003); 11. Nat Biotechnol 21, 1387 (2003). A number of references describe the biological properties of some quantum dots used in experiments. These papers are selected to represent some of the different classes of applications, but this list is not exhaustive. These materials are all quite different from the Qdot conjugates that are sold by Invitrogen, and the results are not necessarily representative of results attainable with these materials: 12. Science 281, 2013 (1998); 13. Science 281, 2016 (1998); 14. J Am Chem Soc 124, 4586 (2002); 15. Proc Natl Acad Sci U S A. 99, (2002); 16. Science 298, 1759 (2002); 17. Nat Biotechnol 21, 41 (2003); 18. Nat Biotechnol 21, 47 (2003). Also of interest: 19. Lakowicz, J. Principles of Fluorescence Spectroscopy. Kluwer Academic Publishing, 1999; 20. Conf Proc IEEE Eng Med Biol Soc 1, 1470 (2006); 21. J Fluoresc 17, 193 (2007); 22. Mol Cell Probes 21, 116 (2006); 23. Kim J, Park, K and Hahn S K, Int J Biol Macromol, in press (2007); 24. Stem Cells 25, 2128 (2007); 25. Hotz, CZ., and Bruchez, M. Quantum Dots: Applications in Biology (Methods in Molecular Biology) J Am Chem Soc 128, (2006). Qdot ITK Carboxyl Quantum Dots 10

11 Prod uct List Current pric es may be ob tained from our website or from our Customer Service Department. Cat no. Product Name Unit Size Q21341MP Qdot 525 ITK carboxyl quantum dots *8 µm solution* µl Q21391MP Qdot 545 ITK carboxyl quantum dots *8 µm solution* µl Q21331MP Qdot 565 ITK carboxyl quantum dots *8 µm solution* µl Q21311MP Qdot 585 ITK carboxyl quantum dots *8 µm solution* µl Q21301MP Qdot 605 ITK carboxyl quantum dots *8 µm solution* µl A10200 Qdot 625 ITK carboxyl quantum dots *8 µm solution* µl Q21321MP Qdot 655 ITK carboxyl quantum dots *8 µm solution* µl Q21361MP Qdot 705 ITK carboxyl quantum dots *8 µm solution* µl Q21371MP Qdot 800 ITK carboxyl quantum dots *8 µm solution* µl Related products S888 Streptavidin mg Contact Information Molecular Probes, Inc Willow Creek Road Eugene, OR Phone: (541) Fax: (541) Customer Service: 6:00 am to 4:30 pm (Pacific Time) Phone: (541) Fax: (541) probesorder@invitrogen.com Toll-Free Ordering for USA: Order Phone: (800) Order Fax: (800) Technical Service: 8:00 am to 4:00 pm (Pacific Time) Phone: (541) Toll-Free (800) Fax: (541) probestech@invitrogen.com Invitrogen European Headquarters Invitrogen, Ltd. 3 Fountain Drive Inchinnan Business Park Paisley PA4 9RF, UK Phone: +44 (0) Fax: +44 (0) euroinfo@invitrogen.com Technical Services: eurotech@invitrogen.com Further information on Molecular Probes products, including product bibliographies, is available from your local distributor or directly from Molecular Probes. 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